A Robust Platform for the Synthesis of New Tetracycline ......A. G. Science 2005, 308, 395–398....

A Robust Platform for the Synthesis of New TetracyclineAntibiotics

Cuixiang Sun, Qiu Wang, Jason D. Brubaker, Peter M. Wright, Christian D. Lerner,Kevin Noson, Mark Charest, Dionicio R. Siegel, Yi-Ming Wang, and

Andrew G. Myers*

Department of Chemistry and Chemical Biology, HarVard UniVersity,Cambridge, Massachusetts 02138

Received August 21, 2008; E-mail: [email protected]

Abstract: Tetracyclines and tetracycline analogues are prepared by a convergent, single-step Michael-Claisencondensation of AB precursor 1 or 2 with D-ring precursors of wide structural variability, followed by removalof protective groups (typically in two steps). A number of procedural variants of the key C-ring-formingreaction are illustrated in multiple examples. These include stepwise deprotonation of a D-ring precursorfollowed by addition of 1 or 2, in situ deprotonation of a D-ring precursor in mixture with 1 or 2, and in situlithium-halogen exchange of a benzylic bromide D-ring precursor in the presence of 1 or 2, followed bywarming. The AB plus D strategy for tetracycline synthesis by C-ring construction is shown to be robustacross a range of different carbocyclic and heterocyclic D-ring precursors, proceeding reliably and with ahigh degree of stereochemical control. Evidence suggests that Michael addition of the benzylic anion derivedfrom a given D-ring precursor to enones 1 or 2 is quite rapid at -78 °C, while Claisen cyclization of theenolate produced is rate-determining, typically occurring upon warming to 0 °C. The AB plus D couplingstrategy is also shown to be useful for the construction of tetracycline precursors that are diversifiable bylatter-stage transformations, subsequent to cyclization to form the C ring. Results of antibacterial assaysand preliminary data obtained from a murine septicemia model show that many of the novel tetracyclinessynthesized have potent antibiotic activities, both in bacterial cell culture and in vivo. The platform fortetracycline synthesis described gives access to a broad range of molecules that would be inaccessible bysemisynthetic methods (presently the only means of tetracycline production) and provides a powerful enginefor the discovery and, perhaps, development of new tetracycline antibiotics.

Introduction

In prior research we showed that cyclohexenones 1 and 2can be transformed into 6-deoxytetracycline antibiotics using asequence of as few as three chemical steps.1

The first and key step of the sequence forms the C ring ofthe tetracyclines by a Michael-Claisen cyclization reaction, apotentially general means for constructing tetracycline analogues

widely variant in the left-hand or D-ring portion of themolecule.2 Here, we expand upon our original findings, describ-ing the synthesis of more than 50 different tetracyclines andtetracycline analogues, many of which are active in inhibitingthe growth of cultured Gram-positive and Gram-negativebacteria, including tetracycline-resistant strains. We providedetailed protocols for the key cyclization reaction in its variousforms and discuss features of stereochemistry, chemical ef-ficiency, and mechanism. We also report minimum inhibitoryconcentration values for selected analogues in a number ofdifferent bacterial strains, as well as preliminary in vivo dataobtained in a mouse septicemia model using a tetracycline-sensitive strain of Staphylococcus aureus.

Background

The first tetracycline antibiotic was discovered in 1948, whenBenjamin Duggar of Lederle Laboratories isolated the naturalproduct chlorotetracycline (Aureomycin, 3) from the culturebroth of a novel species of Streptomyces.3 Within two years, aresearch team from Chas. Pfizer and Co. had isolated a secondnatural tetracycline, oxytetracycline (Terramycin, 4),4 and in

(1) Charest, M. G.; Lerner, C. D.; Brubaker, J. D.; Siegel, D. R.; Myers,A. G. Science 2005, 308, 395–398.

(2) As pointed out by a reviewer, the second step of the key C-ring-formingreaction sequence is more properly designated as an internal Claisenreaction, rather than a Dieckmann reaction, as we previously describedit; for discussion, see: (a) Hauser, C. R.; Swamer, F. W.; Adams, J. T.Org. React. (N.Y.) 1954, 8, 59. (b) Schaefer, J. P.; Bloomfield, J. J.Org. React. (N.Y.) 1967, 15, 1. For brevity, this sequence is referredto here as a Michael-Claisen cyclization.

(3) (a) Duggar, B. M Ann. N.Y. Acad. Sci. 1948, 51, 177–181. (b) Duggar,B. M. Aureomycin and Preparation of Same. U.S. Patent 2,482,055,Sept 13, 1949.

Published on Web 12/03/2008

10.1021/ja806629e CCC: $40.75 2008 American Chemical Society J. AM. CHEM. SOC. 2008, 130, 17913–17927 9 17913

1953, tetracycline itself (5) was prepared from chlorotetracyclineby catalytic hydrogenolysis of the carbon-chlorine bond, atransformation discovered by Lloyd Conover of Pfizer.5 Sub-sequently, tetracycline was found to be a natural product,6 andlater still, Lederle researchers isolated 6-demethyltetracyclines(see structure 6) from culture broths of a mutant strain ofStreptomyces.7 Conover has provided detailed and insightfulaccounts of research efforts leading to new tetracyclinesspecifically and antibiotics more generally.8 All tetracyclinesapproved for human or veterinary use are fermentation productsor are derived from fermentation products by semisynthesis.This is also true of most �-lactam and all macrolide antibiotics.Tracing the paths of human efforts to produce new antibioticsfrom natural products not accessible by synthesis reveals anevolutionary process marked by specific, impactful discoveries.In the case of the tetracyclines, Pfizer scientists achieved a majorenabling advance approximately 10 years after the class hadbeen identified when they demonstrated that the C6-hydroxylgroup of the natural products oxytetracycline (4), tetracycline(5), and 6-demethyltetracycline (6) could be removed reduc-tively.9 The 6-deoxytetracyclines produced, including 6-deox-ytetracycline itself (7), were found to be more stable than theparent compounds, yet they retained broad-spectrum antibacte-rial activity. The important and now generic antibiotics doxy-cycline (8; Pfizer, 1967) and minocycline (9; Lederle, 1972)followed as a consequence, the latter arising from the additionaldiscovery that electrophilic aromatic substitution at C7 becomespossible when the more stable 6-deoxytetracyclines are usedas substrates.10 Decades later, a team of Wyeth scientists ledby Frank Tally synthesized 7,9-disubstituted tetracycline deriva-tives, leading to the discovery of the antibiotic tigecycline(Tigacyl, 10; U.S. approval 2005).11

Tigacyl is one of only three new antibiotics to be brought tomarket in the United States in the past three years, and the onlybroad-spectrum agent.12 Diminishing economic incentives andincreasing regulatory hurdles have led many pharmaceuticalcompanies to discontinue efforts to develop new antibiotics,

raising concern among public health officials, particularly withthe emergence of antibiotic-resistant bacterial strains in com-munity settings. While careful management of the use of existingantibiotics in society is warranted, it would seem unwise toabandon the search for new agents, given the diversity ofbacteria and their capacity to evolve rapidly. Attempts to developantibacterial agents with novel targets have met with littlesuccess.13 As a consequence, many research programs seekingto discover new antibiotics have been refocused toward themodification of agents in proven classes, such as the tetracy-clines, with an emphasis on overcoming bacterial resistance.While the innovations of chemists seeking to modify thestructure of naturally occurring tetracyclines have been extraor-dinary, the slowing pace of discovery in this area is evident.

From the time that the structures of the tetracycline antibioticswere first revealed by Woodward and collaborators,14 manylaboratories have sought to develop a practical route for theirsynthesis. In 2003, an expert opinion of the patent literaturesummarized the state of the art, concluding, “the original effortof Woodward has survived as the basic strategy for the totalsynthesis of this series and at greater than 25 steps is clearlynot to be considered as practical. ... we believe there is amplejustification to explore new total synthetic and convergentsynthetic routes that take full advantage of the body of chemistrythat has become available since Woodward’s original effort.” 15

Among the many developments since Woodward and co-workers first synthesized sancycline (6-deoxy-6-demethyltetra-

(4) (a) Finlay, A. C.; Hobby, G. L.; P’an, S. Y.; Regna, P. P.; Routien,J. B.; Seeley, D. B.; Shull, G. M.; Sobin, B. A.; Solomons, I. A.;Vinson, J. W.; Kane, J. H. Science 1950, 111, 85. (b) Sobin, B. A.;Finlay, A. C. Terramycin and its Production. U.S. Patent 2,516,080,July 18, 1950.

(5) (a) Booth, J. H.; Morton, J.; Petisi, J. P.; Wilkinson, R. G.; Williams,J. H. J. Am. Chem. Soc. 1953, 75, 4621. (b) Conover, L. H.; Moreland,W. T.; English, A. R.; Stephens, C. R.; Pilgrim, F. J. J. Am. Chem.Soc. 1953, 75, 4622–4623.

(6) Minieri, P. P.; Sokol, H.; Firman, M. C. Process for the Preparationof Tetracycline and Chlorotetracycline. U.S. Patent 2,734,018, Feb 7,1956.

(7) McCormick, J. R. D.; Sjolander, N. O.; Hirsch, U.; Jensen, E. R.;Doerschuk, A. P. J. Am. Chem. Soc. 1957, 79, 4561–4563.

(8) (a) Conover, L. H. Res. Technol. Management 1984, 17–22. (b)Conover, L. H. ACS AdV. Chem. Ser. 1971, 108, 33–80.

(9) (a) Stephens, C. R.; Murai, K.; Rennhard, H. H.; Conover, L. H.;Brunings, K. J. J. Am. Chem. Soc. 1958, 80, 5324–5325. (b)McCormick, J. R. D.; Jensen, E. R.; Miller, P. A.; Doerschuk, A. P.J. Am. Chem. Soc. 1960, 82, 3381–3386. (c) Stephens, C. R.;Beereboom, J. J.; Rennhard, H. H.; Gordon, P. N.; Murai, K.;Blackwood, R. K.; Schach von Wittenau, M. J. Am. Chem. Soc. 1963,85, 2643–2652. (d) Blackwood, R. K.; Stephens, C. R. J. Am. Chem.Soc. 1962, 84, 4157–4159.

(10) (a) Martell, M. J.; Boothe, J. H. J. Med. Chem. 1967, 10, 44–46. (b)Church, R. F. R.; Schaue, R. E.; Weiss, M. J. J. Org. Chem. 1971,36, 723–725. (c) Spencer, J. L.; Hlavka, J. J.; Petisi, J.; Krazinski,H. M.; Boothe, J. H. J. Med. Chem. 1963, 6, 405–407. (d) Zambrano,R. T. U.S. Patent 3,483,251, Dec 9, 1969.

(11) (a) Sum, P.-E.; Lee, V. J.; Testa, R. T.; Hlavka, J. J.; Ellestad, G. A.;Bloom, J. D.; Gluzman, Y.; Tally, F. P. J. Med. Chem. 1994, 37, 184–188. (b) Sum, P.-E.; Petersen, P. Bioorg. Med. Chem. Lett. 1999, 9,1459–1462.

(12) Listing of Newly Approved Drug Therapies (2008)http://www.centerwatch.com/patient/drugs/druglist.html, 2008 (accessed August2008).

(13) Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Pompliano, D. L. Nat.ReV. Drug DiscoVery 2007, 6, 29–40.

(14) (a) Hochstein, F. A.; Stephens, C. R.; Conover, L. H.; Regna, P. P.;Pasternack, R.; Gordon, P. N.; Pilgrim, F. J.; Brunings, K. J.;Woodward, R. B. J. Am. Chem. Soc. 1953, 75, 5455–5475. (b)Stephens, C. R.; Conover, L. H.; Hochstein, F. A.; Regna, P. P.;Pilgrim, F. J.; Brunings, K. J. J. Am. Chem. Soc. 1952, 74, 4976–4977.

(15) Podlogar, B. L.; Ohemeng, K. A.; Barrett, J. F. Expert Opin. Ther.Patents 2003, 13, 467–478.

17914 J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008

A R T I C L E S Sun et al.

cycline) in 1962,16 one of particular note, and of relevance tothe results described herein, is Stork and Hagedorn’s strategyfor protection of the vinylogous carbamic acid group of the Aring of the tetracyclines as a 3-benzyloxyisoxazole group;subsequent deprotection occurs under mild (hydrogenolytic)conditions.17

Strategically, the original route developed by Woodward andcollaborators for the synthesis of sancycline employed a “left-to-right” or DfA mode of construction. The Shemyakin andMuxfeldt research groups adopted a similar directionality in theirremarkable syntheses of tetracycline (5; 1967) and terramycin(4; 1968), respectively, using a bicyclic CD-ring precursor asstarting material.18,19 With the benefit of more than 50 years ofstructure-activity relationship data, as well as X-ray crystalstructures of tetracycline bound to the bacterial ribosome (itsputative target),20 the left-to-right mode of construction usedin these pioneering synthetic efforts can be seen to present apractical disadvantage to the discovery of new tetracyclineantibiotics, for the D ring has emerged as one of the mostpromising sites for structural variation. This was a primaryconsideration guiding our initial retrosynthetic analysis of thetetracycline class, leading us to focus upon disconnection ofthe C ring. Thus, we envisioned assembling tetracyclines by aconvergent coupling of D- and AB-ring precursors. Althoughmodel studies suggested that both Diels-Alder and Michael-Claisen cyclization reactions might be used to form the C ring,21

only the latter proved successful with the AB precursors thatwe later targeted and synthesized (1 and 2).

Michael-Claisen and Michael-Dieckmann reaction se-quences have been widely employed to construct naphthalenederivatives since 1978, when three different cyclization protocolswere introduced by independent research groups. Hauser andRhee used a sulfoxide-stabilized o-toluate ester anion as thenucleophilic component in a Michael-Dieckmann cyclizationreaction with methyl crotonate (eq 1). In this case, aromatizationoccurred upon thermal elimination of phenylsulfenic acid.22 Theuse of phthalide and cyanophthalide anions as nucleophiliccomponents was described by Broom and Sammes (eq 2)23 andKraus and Sugimoto (eq 3),24 respectively. Formal loss of waterand hydrogen cyanide, respectively, led to naphthoate esterproducts in these procedures. In 1979, the Weinreb and Staunton

research groups first reported that simple o-toluate ester anions(unsubstituted at the benzylic position) undergo Michael-Claisencyclization reactions with �-methoxycyclohexenones and γ-py-rones to form naphthyl ketones (see eq 4 for one example), asequence sometimes referred to as the Staunton-Weinrebannulation.25

There is also precedent for the formation of non-aromaticsix-membered rings by Michael-Claisen and Michael-Dieckmann reaction sequences.23,26,27 With few exceptions,27

stereochemical features of these cyclization reactions have notbeen discussed, frequently because they were of little conse-quence (aromatization followed cyclization). The Michael-Claisencyclizations detailed below are unusual in their stereochemicalcomplexity, stereocontrol, and efficiency. In 2000, while ourstudies were in progress, Tatsuta and co-workers reported asynthesis of (-)-tetracycline (34 steps, 0.002% yield) thatemployed an early stage Michael-Claisen cyclization reactionto form an aromatic C-ring precursor, which was dearomatizedlater in the sequence.28 Since 2005, our laboratory has reportedtwo different routes to synthesize the AB precursor 1 in opticallyactive form; the more recent of these was scaled to prepare >40g of crystalline product in one batch.29 Here we provide detailsof the different protocols that can be used to construct the C

(16) (a) Conover, L. H.; Butler, K.; Johnston, J. D.; Korst, J. J.; Woodward,R. B. J. Am. Chem. Soc. 1962, 84, 3222–3224. (b) Woodward, R. B.Pure Appl. Chem. 1963, 6, 561–573. (c) Korst, J. J.; Johnston, J. D.;Butler, K.; Bianco, E. J.; Conover, L. H.; Woodward, R. B. J. Am.Chem. Soc. 1968, 90, 439–457.

(17) Stork, G.; Hagedorn, A. A., III. J. Am. Chem. Soc. 1978, 100, 3609–3611.

(18) (a) Gurevich, A. I.; Karapetyan, M. G.; Kolosov, M. N.; Korobko,V. G.; Onoprienko, V. V.; Popravko, S. A.; Shemyakin, M. M.Tetrahedron Lett. 1967, 8, 131–134. (b) Kolosov, M. N.; Popravko,S. A.; Shemyakin, M. M. Liebigs Ann. 1963, 668, 86–91.

(19) (a) Muxfeldt, H.; Hardtmann, G.; Kathawala, F.; Vedejs, E.; Mooberry,J. B. J. Am. Chem. Soc. 1968, 90, 6534–6536. (b) Muxfeldt, H.; Haas,G.; Hardtmann, G.; Kathawala, F.; Mooberry, J. B.; Vedejs, E. J. Am.Chem. Soc. 1979, 101, 689–701.

(20) (a) Brodersen, D. E.; Clemons, W. M., Jr.; Carter, A. P.; Morgan-Warren, R. J.; Wimberly, B. T.; Ramakrishnan, V. Cell 2000, 103,1143–1154. (b) Pioletti, M.; Schlunzen, F.; Harms, J.; Zarivach, R.;Gluhmann, M.; Avila, H.; Bashan, A.; Bartels, H.; Auerbach, T.;Jacobi, C.; Hartsch, T.; Yonath, A.; Franceschi, F EMBO J. 2001, 20,1829–1839.

(21) Parrish, C. A. Ph.D. Thesis, California Institute of Technology,Pasadena, CA, 1999.

(22) Hauser, F. M.; Rhee, R. P. J. Org. Chem. 1978, 43, 178–180.(23) Broom, N. J. P.; Sammes, P. G. J. Chem. Soc., Chem. Commun. 1978,

162–164.(24) Kraus, G. A.; Sugimoto, H. Tetrahedron Lett. 1978, 26, 2263–2266.

(25) (a) Dodd, J. H.; Weinreb, S. M. Tetrahedron Lett. 1979, 38, 3593–3596. (b) Dodd, J. H.; Starrett, J. E.; Weinreb, S. M. J. Am. Chem.Soc. 1984, 106, 1811–1812. (c) Leeper, F. J.; Staunton, J. J. Chem.Soc., Chem. Commun. 1979, 5, 206–207. (d) Leeper, F. J.; Staunton,J. J. Chem. Soc., Perkin Trans. 1 1984, 1053–1059.

(26) (a) Tarnchompoo, B.; Thebtaranonth, C.; Thebtaranonth, Y. Synthesis1986, 9, 785–786. (b) Boger, D. L.; Zhang, M. J. Org. Chem. 1992,57, 3974–3977. (c) Nishizuka, T.; Hirosawa, S.; Kondo, S.; Ikeda,D.; Takeuchi, T. J. Antibiot. 1997, 50, 755–764. (d) Hill, B.; Rodrigo,R. Org. Lett. 2005, 7, 5223–5225.

(27) For examples of stereocontrol in Michael-Claisen and Michael-Dieckmann cyclization reactions, see: (a) Franck, R. W.; Bhat, V.;Subramaniam, C. S. J. Am. Chem. Soc. 1986, 108, 2455–2457. (b)Tatsuta, K.; Yamazaki, T.; Mase, T.; Yoshimoto, T. Tetrahedron Lett.1998, 39, 1771–1772. (c) White, J. D.; Demnitz, F. W. J.; Qing, X.;Martin, W. H. C. Org. Lett. 2008, 10, 2833–2836.

(28) Tatsuta, K.; Yoshimoto, T.; Gunji, H.; Okado, Y.; Takahashi, M. Chem.Lett. 2000, 646–647.

(29) Brubaker, J. D.; Myers, A. G. Org. Lett. 2007, 9, 3523–3525.

J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008 17915

Synthesis of New Tetracycline Antibiotics A R T I C L E S

ring of the tetracyclines and exemplify these protocols with thepreparation of a number of novel substances with antibacterialproperties.

Results

A critical early experiment in our attempts to assemble theC ring of the tetracyclines by a Michael-Claisen cyclizationreaction provided both direction and mechanistic insight. Asdepicted in Scheme 1, treatment of a solution of organostannane1130 in tetrahydrofuran (THF) with n-butyllithium at -78 °C,followed by transfer of this mixture to a solution of enone 1,also at -78 °C, and subsequent quenching with tert-butyl-dimethylsilyl triflate (TBSOTf), afforded the Michael additionproduct 12 as a single stereoisomer in 98% yield.

Crystallization of the chromatographically purified productfrom hexanes at -30 °C provided a single crystal suitable forX-ray analysis (mp 146 °C). Inspection of the crystal structure(Figure 1) revealed that the stereochemistry of positions “C5a”and “C6” of the adduct 12 corresponded to that of 6-deoxytet-racycline (7) and doxycycline (8), which was fortuitous giventhat as many as four diastereomers could have been formed inthe Michael addition reaction. The product that is formedapparently arises from selective addition of the nucleophile tothe concave face of the enone. This selectivity may arise as aconsequence of stereoelectronic factors (pseudoaxial addition

to the enone) and/or steric effects (addition of the nucleophileto the π-face opposite the tert-butyldimethylsilyloxy substituent,which is also axial; see the space-filling model of enone 1depicted in Figure 1).

Directed by this key finding, we proceeded to strategicallymodify the D-ring precursor, with three objectives: (1) to activatethe ester toward Claisen cyclization (which we did not observewith the methyl ester substrate 11), (2) to obviate the use oforganotin intermediates, and (3) to mask the C10 phenoxysubstituent with a protective group more labile than methyl.These objectives were achieved using the tert-butoxycarbonyl-protected phenyl ester substrate 13 (Scheme 2).31,32

Deprotonation of 13 (3 equiv) with lithium diisopropylamide(LDA, 4 equiv) at -78 °C in the presence of N,N,N′,N′-tetramethylethylenediamine (TMEDA, 4 equiv) afforded a deepred solution of the corresponding o-toluate ester anion; additionof a solution of the enone 1 (1 equiv) and slow warming of theresulting mixture to 0 °C over 3 h provided the Michael-Claisencyclization product 14 in 81% yield in diastereomerically pureform after isolation by reverse-phase high-performance liquidchromatography (rp-HPLC). A minor diastereomer (<4%),believed to be epimeric at C6, was isolated separately.

Thus, Michael addition occurs with >20:1 stereoselectivityat C6, in the sense indicated in Scheme 2, and appears to proceedwith complete stereocontrol at C5a (attack upon a singlediastereoface of the enone). These observations have heldconsistently in more than 40 different C-ring-forming cyclizationreactions examined to date, with two (closely related) exceptions,discussed below.

There is little question that the transformation of enone 1 tothe 6-deoxytetracycline precursor 14 proceeds by a stepwisemechanism, involving sequential Michael and Claisen reactions,for the intermediate Michael adduct can be intercepted byprotonation with acetic acid at -78 °C to give the keto ester16 in 88% yield (eq 5).33

Scheme 1. Michael Addition of a Benzylic Anion (Generated byTin-Lithium Exchange) to Enone 1, Followed by Trapping of theResultant Enolate with tert-Butyldimethylsilyl Triflate

Figure 1. Crystal structures of the Michael addition product 12 and theenone 1, presented as ball and stick and space-filling models, respec-tively.

Scheme 2. Synthesis of 6-Deoxytetracycline (7) byMichael-Claisen Cyclization Using thetert-Butoxycarbonyl-Protected Phenyl Ester 13 as the D-RingPrecursor and a Stepwise Protocol for Addition of the Base andEnone 1, Followed by Deprotection



Indeed, we have observed across a range of different D-ringprecursors that Michael addition is relatively rapid at -78 °C,while Claisen cyclization proceeds more slowly, typically uponwarming to 0 °C. Thus, we view Claisen cyclization and notMichael addition as rate-determining. It is evident from theseobservations that C-ring formation cannot occur by Diels-Aldercycloaddition of an o-quinonemethide intermediate, which ishypothetically a mechanistic alternative to Michael-Claisencyclization.

With the establishment of an effective protocol for construc-tion of the C ring, a two-step deprotection sequence wasemployed to transform the cyclization product 14 into 6-deox-ytetracycline (Scheme 2). The tert-butoxycarbonyl and tert-butyldimethylsilyl protective groups were removed upon treat-ment of 14 with hydrofluoric acid in acetonitrile at 23 °C (2days). Hydrogenolysis of the crude reaction product (15) in thepresence of a palladium catalyst under an atmosphere ofhydrogen in methanol-dioxane at 23 °C and subsequentpurification by rp-HPLC then afforded 6-deoxytetracycline (7)in 85% yield.17 As the examples below will demonstrate, thistwo-step deprotection protocol has been found to be widelyapplicable, though in some instances it is advantageous to invertthe ordering of the two steps. In general, the tert-butoxycarbonylgroup is cleaved relatively rapidly in the deprotection stepemploying hydrofluoric acid, while the tertiary tert-butyldim-ethylsilyl ether undergoes protodesilylation more slowly.

The conditions developed for the cyclization reaction depictedin Scheme 2, sequential deprotonation of a D-ring precursorfollowed by addition of the enone 1, have been effective forthe synthesis of a number of known tetracyclines as well asnovel tetracycline analogues variant within or near the D ring.For example, treatment of phenyl ester 1734 (3 equiv, Scheme3) with LDA (3 equiv) in the presence of TMEDA (6 equiv) at-78 °C, followed by addition of enone 1 (1 equiv) and warmingof the resulting mixture to -10 °C, provided the Michael-Claisencyclization product 18 in 83% yield after purification by rp-HPLC. Two-step deprotection and purification by rp-HPLC thenafforded minocycline (9) in 74% yield.

Among the modified D-ring precursors we have investigated,substrates with benzylic anion-stabilizing groups were foundto undergo particularly efficient cyclization reactions. Forexample, addition of enone 1 (1 equiv) to a solution of thestabilized anion derived from substrate 19 (3 equiv, Scheme

4),32 followed by warming of the resulting mixture to -15 °C,provided the Michael-Claisen cyclization product 20 in 97%yield after purification by rp-HPLC. Standard deprotection of20 then afforded the novel tetracycline analogue 6-(S)-phenyl-sancycline (21), which was found to effectively inhibit thegrowth of a number of Gram-positive bacteria, includingtetracycline-resistant strains (vide infra), prompting the synthesisof a series of 6-aryl-substituted tetracyclines (see Table 1).35 Itis worthy of note that more than half of the cyclization reactionspresented in the tables below were attempted only once.

Other notable features of the cyclization reactions of D-ringprecursors with benzylic anion-stabilizing groups include thefact that additives such as TMEDA were typically not requiredand that stoichiometries of just over 1 equiv of a given D-ringprecursor frequently sufficed to achieve a high yield of cycliza-tion product. We also found that benzylic deprotonation couldbe conducted with the weaker base lithium bis(trimethylsilyl)-amide (LHMDS) in lieu of the standard base LDA, and, usingN-imidazoyl substrate 22 (Scheme 5), the important observationwas made that condensation could be achieved with highefficiency by an in situ deprotonation protocol, which is to sayby addition of base to the D-ring precursor in the presence of

(30) Carpenter, T. A.; Evans, G. E.; Leeper, F. J.; Staunton, J.; Wilkinson,M. R. J. Chem. Soc., Perkin Trans. I 1984, 1043–1051.

(31) Phenyl ester activation in toluate ester condensations is precedented:(a) White, J. D.; Nolen, E. G., Jr.; Miller, C. H. J. Org. Chem. 1986,51, 1150–1152 (see also ref 27c).

(32) Bennetau, B.; Mortier, J.; Moyroud, J.; Guesnet, J.-L. J. Chem. Soc.,Perkin Trans. 1 1995, 1265–1271.

(33) For other examples of the isolation of uncyclized Michael adducts,see refs 23, 25c, 25d, and 26b.

(34) Phenyl ester 17 was synthesized in six steps from ethyl 6-methylsali-cylate; see: (a) Olah, G. A.; Malhotra, R.; Narang, S. C. Nitration,Methods and Mechanisms; VCH Publishers Inc.: New York, 1980.(b) Bellamy, F. D.; Ou, K. Tetrahedron Lett. 1984, 26, 839–842. (c)Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.;Shah, R. D. J. Org. Chem. 1996, 61, 3849–3862.

(35) Leading references for the synthesis of D-ring precursors to 6-aryl-substituted tetracyclines: (a) Miyaura, N.; Suzuki, A Chem. ReV. 1995,95, 2457–2483. (b) Kotha, S.; Mandal, K. Eur. J. Org. Chem. 2006,23, 5387–5393. (c) Wright, S. W.; Hageman, D. L.; McClure, L. D.J. Org. Chem. 1994, 59, 6095–6097. (d) Osyanin, V. A. ; Purygin,P. P.; Belousova, Z. P. Russ. J. Gen. Chem. 2005, 75, 111–117. (e)Appukkuttan, P.; Dehaen, W.; Fokin, V. V.; Van der Eycken, E. Org.Lett. 2004, 6, 4223–4225.

Scheme 3. Synthesis of Minocycline (9) by Michael-ClaisenCyclization Using the tert-Butoxycarbonyl-Protected Phenyl Ester17 as the D-Ring Precursor and a Stepwise Protocol for Additionof the Base and Enone 1, Followed by Deprotection

Scheme 4. Synthesis of 6-(S)-Phenylsancycline (21) byMichael-Claisen Cyclization Using thetert-Butoxycarbonyl-Protected Phenyl Ester 19 as the D-RingPrecursor and a Stepwise Protocol for Addition of the Base andEnone 1, Followed by Deprotection



Table 1. Synthesis of 6-Substituted Tetracyclines by Michael-Claisen Cyclization Using D-Ring Precursors with Anion-StabilizingSubstituents in the Benzylic Position and a Stepwise Protocol for Addition of the Base and Enone 1, Followed by Deprotection [(i) HF (aq),CH3CN; (ii) H2, Pd/C (or Pd black), CH3OH-dioxane]



enone 1. Thus, treatment of a mixture of phenyl ester 22 (1.3equiv) and enone 1 (1 equiv) with an excess of LHMDS (3.3equiv) at -78 °C, followed by warming of the resulting mixtureto -30 °C over 90 min, provided the cyclization product 23 in94% yield (Scheme 5); removal of protective groups thenprovided 6-(R)-N-imidazoylsancycline (24, 42% yield over twosteps).

In situ deprotonation with LHMDS was also effective inbringing about cyclization of the methyl phenyl diester substrate25 with enone 1, as well as cyclization of the correspondingbenzyl phenyl diester substrate 27, as depicted in Scheme 6. Inboth cases, the major product of cyclization was epimeric atC6 relative to the major products of all other cyclization

reactions we have studied (these providing the two exceptionsreferred to above), which we attribute to epimerization at C6after cyclization. We have not yet conducted the rigorous studiesnecessary to establish if the product ratios were kinetically orthermodynamically determined in these examples.

The in situ deprotonation protocol has proven to be effectivefor the Michael-Claisen cyclization reactions of a number ofdifferent D-ring precursors containing benzylic anion-stabilizingsubstituents (see Table 2). Because of its greater experimentalconvenience, this has become the preferred method for thesynthesis of tetracycline analogues substituted at the C6-position.

D-ring heterocyclic analogues of tetracyclines had not beenmade before, so far as we are aware, and their construction bysemisynthesis cannot be easily imagined. In targeting analoguesof this type, we initially chose to explore the synthesis of theD-ring pyridine derivative 7-aza-10-deoxysancycline (30, Scheme7). As we previously reported, in situ deprotonation of phenylester 28 (4 equiv) with LDA (5 equiv) at -95 °C in the presenceof enone 1 (1 equiv) and hexamethylphosphoramide (HMPA,10 equiv), followed by warming to -50 °C, afforded theMichael-Claisen cyclization product 29 in 76% yield (Scheme7).1 Claisen ring-closure was notably more facile in this examplethan in others we have studied, proceeding to completion inless than 1 h upon warming to -50 °C. 7-Aza-10-deoxysan-cycline (30) was then obtained in 79% yield after removal ofprotective groups.

Another novel class of tetracyclines that we have exploredis the pentacyclines (Scheme 8). This required that we develop

Scheme 5. Synthesis of 6-(R)-N-Imidazoylsancycline (24) byMichael-Claisen Cyclization Using thetert-Butoxycarbonyl-Protected Phenyl Ester 22 and Enone 1 asSubstrates, with Deprotonation in Situ, Followed by Deprotection

Scheme 6. Synthesis of 6-(S)-Carbomethoxysancycline (26) byMichael-Claisen Cyclization Using the Methyl Phenyl Diester 25and Enone 1 as Substrates, with Deprotonation in Situ, Followedby Deprotectiona

a In one experiment conducted with the corresponding benzyl phenyldiester 27 as the D-ring precursor, a diastereomeric mixture of cyclizationproducts was obtained.

Scheme 7. Synthesis of 7-Aza-10-deoxysancycline (30) byMichael-Claisen Cyclization Using Phenyl Ester 28 and Enone 1as Substrates, with Deprotonation in Situ, Followed byDeprotection

Scheme 8. Synthesis of the Pentacycline 33 by Michael-ClaisenCyclization Using the Phenyl Bromomethylnaphthoic Acid Ester 31and Enone 1 as Substrates, and an in Situ Protocol forLithium-Halogen Exchange, Followed by Deprotection



chemistry suitable for Michael-Claisen cyclization of naph-thoate ester DE-ring precursors,22-24 as the protocols that wehad used to this point were found to be largely ineffective withthese substrates.

Lithium-halogen exchange is rarely employed to formbenzyllithium reagents in organic synthesis, due to the propen-sity of the benzyl halide substrates to engage in Wurtz-typecoupling reactions in the presence of an alkyllithium reagent.36

Table 2. Synthesis of 6-Substituted Tetracyclines by Michael-Claisen Cyclization Using D-Ring Precursors with Anion-StabilizingSubstituents in the Benzylic Position and Enone 1 as Substrates, with Deprotonation in Situ, Followed by Deprotection [(i) HF (aq), CH3CN;(ii) H2, Pd/C, CH3OH-dioxane]37



In a surprising transformation, treatment of a solution of phenylbromomethylnaphthoic acid ester 31 (4 equiv) and enone 1 (1equiv) with n-butyllithium (4 equiv) at -100 °C, followed bywarming to 0 °C, provided the Michael-Claisen cyclizationproduct 32 in 75% yield (Scheme 8).1 A three-step deprotectionsequence then afforded pentacycline 33 in 74% yield.

The in situ lithium-halogen exchange protocol developedfor the synthesis of the pentacycline 33 has proven to begenerally effective for the synthesis of a number of quitedifferent tetracycline analogues. In many cases we have adoptedthe procedural modification of substituting the less reactivereagent phenyllithium for n-butyllithium in the lithium-halogenexchange reaction. For example, addition of phenyllithium to asolution of pyrazole 34 (3 equiv) and enone 1 (1 equiv)containing HMPA (6 equiv) at -90 °C, followed by warmingto 0 °C over 2 h, provided the Michael-Claisen cyclizationproduct 35 in 81% yield (Scheme 9). Removal of the protectivegroups led to concomitant cleavage of the heteroaryl carbon-chlorine bond during hydrogenolysis, affording the tetracyclineanalogue 36 containing a D-ring pyrazole (87% yield over twosteps).

A very similar procedure produced 8-fluorosancycline (38)from the benzyl bromide 37 (Scheme 10). Like the D-ringpyrazole analogue 36, the 8-fluorotetracycline analogue 38

(36) (a) Parham, W. E.; Jones, L. D.; Sayed, Y. A. J. Org. Chem. 1978,41, 1184–1186. (b) Berk, S. C.; Yeh, M. C. P.; Jeong, N.; Knochel,P. Organometallics 1990, 9, 3053–3064.

Scheme 9. Synthesis of a Pyrazole Analogue (36) byMichael-Claisen Cyclization Using the Benzylic Bromide 34 andEnone 1 as Substrates, and an in Situ Protocol forLithium-Halogen Exchange, Followed by Deprotection

Scheme 10. Synthesis of 8-Fluorosancycline (38) byMichael-Claisen Cyclization Using Benzylic Bromide 37 andEnone 1 as Substrates, and an in Situ Protocol forLithium-Halogen Exchange, Followed by Deprotection

Scheme 11. Synthesis of 10-Deoxysancycline (41) byMichael-Claisen Cyclization Using Phenyl2-(Bromomethyl)benzoate (39) and Enone 1 as Substrates, withDeprotonation in Situ, Followed by Deprotection

Scheme 12. Synthesis of Pentacyclines with Heterocyclic E Ringsby Michael-Claisen Cyclizations Using Bromomethylquinolinesand Enone 1 as Substrates, and an in Situ Protocol forLithium-Halogen Exchange, Followed by Deprotection



would have been difficult, if not impossible, to prepare bysemisynthetic methods.

Lithium-halogen exchange was also employed for thesynthesis of 10-deoxysancycline (41, Scheme 11). As wereported previously, treatment of a solution of phenyl 2-(bro-momethyl)benzoate (39, 4 equiv) and enone 1 (1 equiv) withn-butyllithium (4 equiv) at -100 °C, followed by warming to

0 °C over 30 min, provided the Michael-Claisen cyclizationproduct 40 in 81% yield.1,38 The typical two-step deprotectionsequence then transformed the cyclized product 40 into 10-deoxysancycline (41) in 84% yield.

Bromomethylquinoline derivatives39 were also employed asDE-ring precursors in Michael-Claisen cyclization reactionswith enone 1, providing pentacycline precursors with hetero-cyclic E rings (Scheme 12). The typical two-step deprotectionsequence afforded tetrahydroquinoline products in these ex-amples, while the use of modified conditions led to deprotectionwithout reduction of the pyridine E-ring. Cyclization reactionsof quinoline substrates with 6-aryl substituents were alsoinvestigated,35b using stepwise deprotonation conditions (Scheme13).

Thus far, each different cyclization reaction described hasproduced a single tetracycline analogue. It is worth notingexplicitly that the convergent coupling strategy employed toprepare individual tetracycline analogues can also be used totarget structures that serve as branch points to large numbersof analogues, versatile structures such as the aryl bromide 43or the aldehyde 44 (Scheme 14). Both of these pentacyclineprecursors were targeted for synthesis.

Michael-Claisen cyclization of the benzylic bromide 4240

withenone1wassuccessfullyachievedbyinsitu lithium-halogenexchange using phenyllithium, but not n-butyllithium. Thus,treatment of a mixture of the bis-bromide 42 (3 equiv) andenone 1 (1 equiv) with phenyllithium (3 equiv) at -100 °C,

(37) The use of a methyl ether protecting group in the penultimate entryof Table 2 required one additional deprotection step (BBr3, CH2Cl2,-78 °C f 23 °C) following the typical two-step sequence.

(38) Both the stepwise and in situ protocols for deprotonation-cyclizationwere also effective for the synthesis of the Michael-Claisen cyclizationproduct 40 from phenyl o-toluate and enone 1, though yields werelower.

(39) Van Leusen, A. M.; Terpstra, J. W. Tetrahedron Lett. 1981, 22, 5097–5100.

(40) Synthesized from 6-bromophthalide and methyl crotonate: Broom,N. J. P.; Sammes, P. G. J. Chem. Soc., Perkin Trans. 1 1981, 465–470.

Scheme 13. Synthesis of 6-Aryl-Substituted HeterocyclicPentacyclines by Michael-Claisen Cyclizations Using BenzylicallySubstituted Quinolines as DE-Ring Precursors and a StepwiseProtocol for Addition of the Base and Enone 1, Followed byDeprotection

Scheme 14. Synthesis of the Diversifiable Pentacycline Precursors43 and 44 by Michael-Claisen Cyclization Using the BenzylicBromide 42 and Enone 1 as Substrates, with an in Situ Protocolfor Selective Lithium-Halogen Exchangea

a Further transformation of 44 by a reductive amination reaction providesa route to alkylaminomethylpentacyclines, as illustrated by the synthesisof the tert-butylaminomethylpentacycline 45.



followed by addition of LHMDS (1 equiv) and warming ofthe resulting mixture to -10 °C, provided the cyclizationproduct 43 in 44% yield (Scheme 14).41 Although the yield

of product in this cyclization reaction was moderate, itremained consistent during scale-up and allowed for sufficientquantities of 43 to be produced to explore late-stagediversification. The aryl bromide intermediate 43 was treatedsequentially with phenyllithium and n-butyllithium to effectdeprotonation and lithium-halogen exchange, respectively,and the resulting dianion was formylated with N,N-dimeth-ylformamide (DMF) to give the aldehyde 44; reductiveamination with tert-butylamine and subsequent deprotectionafforded tert-butylaminomethylpentacycline 45 in 61% yieldafter purification by rp-HPLC. The use of a number ofdifferent amines in the reductive amination reaction led tothe synthesis of various alkylaminomethylpentacycline ana-logues from the common intermediate 44 (Chart 1).

We also successfully pursued the synthesis of a parallelseries of diversifiable pentacycline precursors containing adimethylamino substituent at C7, as in minocycline (9) andtigecycline (10, see Scheme 15). In the case of the dimethy-lamino-substituted naphthoate ester 46, it was possible toconduct cyclization by in situ deprotonation, in the presenceof enone 1, forming the dimethylamino-substituted arylbromide 47 in 57% yield. Subjection of 47 to deprotonation

(41) The yield of the Michael-Claisen cyclization product 43 wassomewhat lower (28%) when LHMDS was omitted from the reaction.

(42) The final compound in Chart 1 was prepared by N-acetylation afterreductive amination with methylamine.

(43) 7-Dimethylamino-alkylaminomethylpentacyclines were initially syn-thesized from a DE-ring precursor in which the phenolic hydroxylgroup was protected as a methyl ether (necessitating a three-stepdeprotection sequence); it was later found that the corresponding DE-ring precursor containing a tert-butoxycarbonyl protecting group(compound 46) was also an effective cyclization substrate (allowingfor standard two-step deprotection, see Supporting Information fordetails).

Chart 1. Alkylaminomethylpentacyclines Synthesized fromAldehyde 44 by Reductive Amination Followed by Deprotectiona

a See Supporting Information for experimental details.42

Scheme 15. Synthesis of the Diversifiable Pentacycline Precursors47 and 48 by Michael-Claisen Cyclization Using the PhenylNaphthoate Ester 46 and Enone 1 as Substrates, withDeprotonation in Situa

a Further transformation of 48 by a reductive amination reaction providesa route to 7-dimethylamino-alkylaminomethylpentacyclines, as illustratedby the synthesis of the 7-dimethylamino-azetidinylmethylpentacycline 49.

Chart 2. 7-Dimethylamino-alkylaminomethylpentacyclinesSynthesized from Aldehyde 48 by Reductive Amination Followedby Deprotection43



and lithium-halogen exchange, and formylation of theresulting dianion with DMF, gave the pentacycline precursoraldehyde 48 in 80% yield; reductive amination with azetidineand subsequent deprotection afforded azetidinylmethylpen-tacycline 49 in 74% yield after purification by rp-HPLC(Scheme 15). As with aldehyde 44 above, reductive aminationof 48 could be conducted using a number of different amines,providing various 7-dimethylamino-alkylaminomethylpenta-cyclines upon deprotection (see Chart 2).

An alternative approach to diversification involved reduc-tion of aldehyde 44 with sodium triacetoxyborohydride,mesylation of the resulting primary alcohol with methane-sulfonic anhydride, and then nucleophilic displacement (seeScheme 16). When imidazole was used as the nucleophile,substitution afforded the N-imidazoylmethyl product 50 (59%yield over two steps); removal of protective groups affordedthe corresponding pentacycline analogue 51 in 40% yield.

As we have previously shown, by variation of our originalsynthetic route to the enone 1 the corresponding C5-benzyl-oxycarbonyloxy substituted enone 2 (see Introduction) can

be prepared in gram amounts.1 This in turn has enabled thesynthesis of a number of C5-hydroxytetracyclines (Scheme17 and Table 3). Like the corresponding cyclization reactionswith enone 1, Michael-Claisen condensations with enone 2proceed with uniformly high stereoselectivity. For example,addition of enone 2 (1 equiv) to a solution of the o-toluateester anion derived from phenyl ester 13 (4.5 equiv) at -78°C, followed by warming of the resulting mixture to 0 °Cover 2 h, provided the Michael-Claisen cyclization product52 in 79% yield in diastereomerically pure form afterpurification by rp-HPLC (Scheme 17). A minor diastereomer,believed to be 6-epi-52, was isolated separately (<7% yield).Doxycycline (8) was obtained in 90% yield after removal ofprotective groups and purification by rp-HPLC.1

Antibacterial Activities

Minimum inhibitory concentrations (MICs) were deter-mined in whole-cell antimicrobial assays using a panel oftetracycline-sensitive and tetracycline-resistant Gram-positiveand Gram-negative bacteria. The results for selected com-

Table 3. Synthesis of 5-Hydroxytetracyclines by Michael-Claisen Cyclization Using a Number of Different D-Ring Precursors and Enone 2as Substrates, Followed by Deprotectiona

a Deprotection conditions: A, (i) HF (aq), CH3CN; (ii) H2, Pd-black, CH3OH-dioxane; B, (i) HF (aq), CH3CN; (ii) H2, Pd/C, CH3OH-dioxane; C,(i) H2, Pd-black, CH3OH-dioxane; (ii) HF (aq), CH3CN.



pounds are shown in Table 4. Two promising new series oftetracycline antibiotics emerge from this study: 6-aryltetra-cyclines, which are active in both tetracycline-sensitive andtetracycline-resistant Gram-positive strains, and alkylami-nomethylpentacyclines, which show good activity in bothtetracycline-sensitive and tetracycline-resistant Gram-positiveand Gram-negative organisms.

Several compounds were selected for further study in a mousesepticemia model to determine efficacy in vivo (Table 5). Micewere inoculated intraperitoneally with an LD90-100 bolus of S.aureus (Smith). One hour later, tetracycline analogues wereadministered intravenously at dose levels ranging from 0.3 to 30

mg/kg. Mortality was monitored once daily for 7 days. All micesurvived at the highest level of dosing (30 mg/kg) for each of thecompounds tested, indicating that the threshold of acute toxicityin mice is greater than this value. The 6-phenyltetracycline analogueshowed some efficacy in this murine model (ED50 10.7 mg/kg),while the alkylaminomethylpentacyclines were more potent, withefficacy similar to that of tetracycline (ED50 1.7-2.8 mg/kg).

Conclusion

The results described illustrate the application of a generalAB plus D strategy for the synthesis of more than 50tetracyclines and tetracycline analogues. C-ring formation was

Table 4. Minimum Inhibitory Concentration (MIC) Values for Selected Tetracycline Analogues (µg/mL)a

a Organisms in red are antibiotic-resistant. Abbreviations: A, ampicillin; M, methicillin; P, penicillin; V, vancomycin; T, tetracycline; Mult,multiple. Organisms: SA, S. aureus; EF, E. faecium; SP, S. pneumoniae; EC, E. coli; AB, A. baumanii; PA, P. aeruginosa; HI, H. influenzae.



achieved by a stereocontrolled Michael-Claisen cyclizationreaction employing one or more of the protocols detailedherein. The examples discussed demonstrate that structural

modification of both AB and D-ring components allows forthe preparation of individual tetracyclines, as well as for thesynthesis of tetracycline precursors that are readily diversifiedby late-stage transformations. In many cases a single cy-clization attempt provided, after deprotection, sufficientmaterial for antibacterial screening against a panel of Gram-positive and Gram-negative organisms. For compounds ofinterest, reactions could then be readily scaled to provideamounts necessary for further evaluation in assays such as a

Table 5. In Vivo Efficacy of Selected Tetracycline Analogues in a Tetracycline-Sensitive Strain of S. aureusa

a Mice were inoculated intraperitoneally with an LD90-100 bolus of S. aureus (Smith) (3.5 × 105 CFU/mouse) in 0.5 mL of BHI brothcontaining 5% mucin. Tetracycline analogues were administered intravenously to test animals at 1 h after bacterial inoculation. Mortality wasmonitored once daily for 7 days.

Scheme 16. An Alternative Route to Diversification of the Aldehyde44, Involving Reduction, Activation, and NucleophilicDisplacement, As Exemplified by the Synthesis of theN-Imidazoylmethylpentacycline 51

Scheme 17. Synthesis of Doxycycline (8) by Michael-ClaisenCyclization Using the tert-Butoxycarbonyl-Protected Phenyl Ester13 as the D-Ring Precursor and a Stepwise Protocol for Additionof the Base and Enone 2, Followed by Deprotection



murine septicemia model. A number of novel structuralclasses were explored, including D-heteroaryl tetracyclines,pentacyclines, and E-heteroaryl pentacyclines. The platformfor tetracycline synthesis described gives access to a broadrange of molecules that would be inaccessible by semisyn-thetic methods (presently the only means of tetracyclineproduction) and provides a powerful engine for the discoveryand, perhaps, development of new tetracycline antibiotics.

Acknowledgment. This work was generously supported bythe National Institutes of Health (AI048825). We thank the NSFfor predoctoral graduate fellowship support (J.D.B. and M.G.C.)and the Deutscher Akademischer Austausch Dienst for postdoc-

toral fellowship support (C.D.L.). We acknowledge Vivisourcefor execution of in vivo studies, Micromyx, LLC for executionof in vitro studies, and Tetraphase Pharmaceuticals for sponsor-ship of these. We thank Dr. Richard Staples and Dr. AndrewHaidle for conducting the X-ray crystallographic analyses.

Supporting Information Available: Detailed experimentalprocedures for Michael-Claisen cyclization reactions anddeprotection sequences, and characterization data for alltetracycline analogues. This material is available free ofcharge via the Internet at http://pubs.acs.org.

JA806629E



A Robust Platform for the Synthesis of New Tetracycline ......A. G. Science 2005, 308, 395–398....

Documents

Transcript of A Robust Platform for the Synthesis of New Tetracycline ......A. G. Science 2005, 308, 395–398....